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Journal of Andrology, Vol. 27, No. 1, January/February 2006Copyright q American Society of Andrology

Semen Parameters in Norwegian Fertile Men

TRINE B. HAUGEN,*† THORE EGELAND,‡ AND ØYSTEIN MAGNUS§

From *Faculty of Health Sciences, Oslo University College; †Andrology Laboratory, Rikshospitalet UniversityHospital; ‡Department of Medical Genetics, Ullevaal University Hospital; and §Department of Gynecology andObstetrics, Rikshospitalet University Hospital, Oslo, Norway.

ABSTRACT: The World Health Organization (WHO) providesguidelines for assessing the various semen variables. A set of ref-erence ranges is given in the WHO Laboratory Manual for the Ex-amination of Human Semen and Sperm-Cervical Mucus Interaction,but several studies indicate that the values should be revised. Fur-thermore, semen parameters obtained at different laboratories arenot directly comparable even if the same methods are used. Thus,it is recommended that each laboratory establish its own referenceranges. In this study, semen from 99 men who had recently achieveda pregnancy were analyzed to establish reference ranges for semenvariables. The reference values were based on the group with timeto pregnancy (TTP) 12 cycles or less (92%) and abstinence timefrom 2 to 7 days. The 5th and 10th percentiles for sperm concentra-tion were 10.6 and 16.9 3 106/mL, respectively, and 33% (5th per-centile) and 43% (10th percentile) for spermatozoa with progressivemotility. These values were below the WHO lower limit. The per-centages of ideal spermatozoa (percentage with normal morphologyaccording to WHO strict criteria) were 3 (5th percentile) and 4 (10th

percentile). Thirty-nine percent reported that their partners becamepregnant during the first cycle after they had stopped using contra-ception. The semen parameters in this group were compared withthe others. Overall, the semen parameters were more favorable inthe group with TTP 5 1 cycle than in the group with TTP . 1. Spermconcentration, progressive motility, and percentage of ideal sper-matozoa according to WHO strict criteria were significantly differentin the 2 groups. However, when analyzed by multiple logistic re-gression, only ‘‘total numbers of sperm with progressive motility’’remained in the model (P 5 .002). This is in accordance with pre-vious studies indicating that a combination of semen characteristicsprovides a better predictor of male fertility potential than the singleparameters. In conclusion, new reference ranges for semen vari-ables deviating from the WHO values are established for our labo-ratory.

Key words: Semen variables, reference range, World Health Or-ganization, partners to pregnant women.

J Androl 2006;27:66–71

Ideally, semen quality should predict the fertility poten-tial for a man. However, the diagnostic value of a se-

men analysis is debated in light of the difficulties in es-tablishing thresholds able to distinguish between fertileand infertile men (reviewed in Tomlinson et al, 1999; vander Merve et al, 2005). Furthermore, semen parametersare considered in different ways on the basis of the clin-ical settings: as part of infertility investigation or follow-up of infertility treatment, for selection for appropriatemethod of assisted reproduction, in reproductive toxicol-ogy, or in contraception studies. Prospective studies onthe association between semen quality and fertility haveshown that sperm concentration or sperm number andsperm morphology have a significant relation to likeli-hood of pregnancy (Bonde et al, 1998; Zinaman et al,2000). Several reports describe differences in semen qual-ity between fertile and subfertile groups (Ombelet et al,1997; Gunalp et al, 2001; Guzick et al, 2001; Menkveld

Correspondence to: Trine B. Haugen, Faculty of Health Sciences, OsloUniversity College, PO Box 4 St Olavs Plass, N-0130 Oslo, Norway (e-mail: trine.b.haugen@hf.hio.no).

Received for publication May 16, 2005; accepted for publication Au-gust 2, 2005.

DOI: 10.2164/jandrol.05010

et al, 2001) in an effort to establish clinical cutoff values.They all showed that at least morphology was a goodpredictor for fertility. In these and other studies the ref-erence ranges for semen variables given in the WorldHealth Organization Laboratory Manual for the Exami-nation of Human Semen and Sperm-Cervical Mucus In-teraction (WHO, 1992, 1999) were discussed. The WHOmanual provides guidelines for assessing the various se-men variables; however, it is still difficult to compare thevalues between laboratories. Furthermore, several studieshave indicated geographical differences in semen quality,probably related to environmental factors; however, ethnicor genetic differences cannot be excluded (Fisch and Go-luboff, 1996; WHO Task Force on Methods of Regulationof Male Fertility, 1996; Jørgensen et al, 2001, 2002; Swanet al, 2003). A common set of reference values may there-fore not be appropriate to use worldwide. In line with thisit is stated in the WHO manual (WHO, 1999) that eachlaboratory should determine its own reference range foreach semen variable. The reference ranges given in thismanual are ‘‘based on the clinical experience of manyinvestigators who have studied populations of healthy fer-tile men.’’ Regarding sperm concentration, total spermcount in the ejaculate, and sperm motility, the referencerange is the same as in the WHO manual from 1992

67Haugen et al · Semen Parameters in Fertile Men

Figure 1. Frequency distribution of time to pregnancy for a group of cou-ples (n 5 97) with pregnant women participating in a study of semenparameters in fertile men.

(WHO, 1992), but an interval for morphology is not giv-en.

For several years the Andrology Laboratory at Riks-hospitalet University Hospital in Oslo used the WHO ref-erence range for comparison of patients’ results. It was,however, obvious that the proportion of Norwegian pa-tients falling outside what was defined as normal rangewas too high, and no appropriate reference range couldbe used in the morphology evaluation. In this study, se-men from partners to pregnant woman was examined toestablish the laboratory’s own reference intervals.

Materials and Methods

Study DescriptionPartners to 500 pregnant women were invited to participate inthe study. The invitation was sent to the woman along with anappointment for a routine screening ultrasound of the fetus (17–18 weeks for a pregnancy) to minimize uncertainty about pater-nity. One hundred and two men accepted the invitation by in-formed consent. The couple filled out a questionnaire about timeto pregnancy (TTP) after the couple had stopped using contra-ceptives and treatment for infertility. Three persons were ex-cluded from the study since the pregnancies were achieved afterassisted reproduction. Each man was given a number upon de-livering the questionnaire, and any identifying personal datawere destroyed. The collected data were stored anonymously.

Semen AnalysisSemen analysis was essentially performed according to WHO(1999) guidelines which are described in detail in the joint Eu-ropean Society of Human Reproduction and Embryology–Nor-dic Association for Andrology (ESHRE-NAFA) manual (Kvistand Bjorndahl, 2002). The Andrology Laboratory, which waspart of the collaboration that resulted in the ESHRE-NAFA man-ual, has been active in international standardization of laboratorymethods for semen analysis and has been a co-organizer of train-ing courses. Furthermore, the laboratory has participated in anexternal quality control program run by the ESHRE Special In-terest Group in Andrology since 1999.

The participants were asked to deliver a semen sample at theday of the ultrasound screening and instructed to keep the ab-stinence time from 2 to 7 days. The abstinence time was re-corded. All the samples were collected in a room close to thelaboratory. For analysis, a spermatozoon without morphologicaldefects as evaluated by the strict WHO criteria (Menkveld et al,1990; WHO, 1999) is defined as ideal (Kvist and Bjorndahl,2002). The teratozoospermia index was based on 4 categories ofdefects according to WHO (1992) and the ESHRE-NAFA man-ual (Kvist and Bjorndahl, 2002). The semen sample was dis-carded after completion of analysis.

Statistical AnalysisStatistical analysis was done using SPSS statistical software forWindows, version 12 (2003) (SPSS Inc, Chicago, Ill). Percen-tiles were calculated using the default option in the SPSS pro-

gram, that is, weighted average. The Mann-Whitney U test wasused to test whether semen variables differed significantly in thegroups defined by TTP 5 1 and TTP . 1. Statistical significancewas defined as P , .05. In addition, multiple regression analysis(logistic and linear) was performed to search for independentpredictor variables. A tolerance interval for a percentile wascomputed in R (http://www.r-project.org/) using bootstrapping.

Results

Age, TTP, and Abstinence TimeThe mean age of the 99 men was 31 years, and the rangespanned from 20 to 45 years. The mean age of their part-ners was 30 years (range, 21–42), and 86% were less than35 years old. The self-reported TTP, recorded as the num-ber of cycles, including the cycle of fertilization, is shownin Figure 1. For 2 persons there was no information onTTP. Thirty-nine percent (38 cases) of the 97 couples withTTP recorded conceived during the first cycle, whereas92% (89 cases) became pregnant within 12 cycles. Whena couple has not conceived within a year, it is usuallyconsidered to be an infertility problem. The distributionof the abstinence time, recorded in days, is shown in Fig-ure 2. The median was 3 days and the mean, 3.7 days.For 3 persons there were no recordings. Ninety-five per-cent of the men had an abstinence time in the range 2 to7 days (Figure 2), which is the recommended abstinence

68 Journal of Andrology · January/February 2006

Figure 2. Distribution of abstinence time in a group of fertile men (n 596).

Table 1. Semen parameters in partners to pregnant women*

Semen Variable

Mean (SD)

Total Group,n 5 99†

Reference Group,n 5 82†

Median (Range)

Total Group,n 5 99†

Reference Group,n 5 82†

Volume (mL)pHSperm concentration (106/mL)Total sperm number (106/ejaculate)

4.2 (2.0)8.3 (0.4)

93.0 (71.4)365.9 (297.9)

3.9 (1.6)8.3 (0.4)

94.0 (71.7)355.8 (272.8)

3.8 (0.7–13.3)8.3 (7.2–10.0)

70.0 (0.9–326.0)291.6 (2.5–1627)

3.7 (0.7–7.6)8.3 (7.2–10.0)

70.0 (0.9–326.0)290.2 (2.5–1380)

Progressive motility (a and b) (%)Rapid progressive motility (a) (%)Ideal spermatozoa (%)

53.3 (8.1)35.0 (11.8)13.6 (7.8)

53.6 (8.3)34.8 (12.0)13.9 (7.6)

55.0 (29.0–72.0)36.0 (10.0–67.0)13.0 (1.0–34.0)

55.0 (29.0–72.0)35.5 (10.0–67.0)13.0 (2.0–34.0)

* Numbers are given for the total group and for a reference group defined by abstinence time 2–7 days and time to pregnancy # 12 cycles. aindicates rapid progressive motility; b, slow or sluggish progressive motility.

† For pH and morphology, n 5 98 and 96 in the total group and n 5 81 and 80 in the reference group.

time interval in the WHO manual (1999). In the follow-ing, the semen parameters from the persons with an ab-stinence time between 2 and 7 days as well as TTP of 12cycles or less were used as basis for establishing referenceranges.

Semen ParametersThe results of semen analysis from the whole study groupare listed in Table 1. The detailed morphology classifi-cation is given in Table 2. When the data were restrictedto those with abstinence time from 2 to 7 days and TTP# 12 cycles, the number of cases was reduced to 82. Thisgroup is referred to as a reference group. The results fromthis group are also shown in Tables 1 and 2. There is onlya slight difference between some of the variables in thetotal and in the reference group. To establish referenceranges for the semen variables, the 2.5th, 5th, and 10thpercentiles are listed, as well as the lower limit of the

WHO (1999) reference ranges (Table 3). For the variousmorphological defects, the 97.5th, 95th, and 90th percen-tiles are shown. It is possible to provide tolerance inter-vals for the percentiles estimated. To illustrate, we haveconsidered the 5th percentile of the ‘‘sperm concentra-tion.’’ We generated 1000 bootstrap samples in R(http://www.r-project.org/), each containing 82 values forsperm concentration. For each sample we calculated the5th percentile. Ninety percent of these values fall in theinterval ranging from 9.0 to 16.5, and this provides a 90%interval for the value 10.6 reported in Table 3.

Association Between Semen Parameters and TTPThe men were divided into 2 groups depending on wheth-er TTP 5 1 cycle or TTP . 1 cycle, and the medianvalues of the semen parameters were compared (Table 4).Overall, the parameters were more favorable in the groupof men in couples who conceived in the first cycle, com-pared to the group that conceived after more than 1 cycle.There were significant differences in sperm concentration,total sperm number, progressive motility, and proportionof ideal spermatozoa. No significant difference was seenin volume, rapid progressive motility, and the varioussperm defects. We performed multiple logistic regressionanalysis. Potential predictor variables were those with P, .1. In addition we controlled for time of abstinence.However, essentially the same results are obtained basingthe analysis on individuals for which abstinence time isrestricted to the interval [2,7] (data not shown). We per-formed backward and forward stepwise logistic regres-sion. In both cases, only the variable ‘‘total number ofsperm with progressive motility’’ remained in the model(P 5 .002).

Discussion

In this study, semen from men who had recently achieveda pregnancy was analyzed to establish reference values

69Haugen et al · Semen Parameters in Fertile Men

Table 2. Morphological defects in the spermatozoa from partners to pregnant women*

Type of Defect

Mean (SD)

Total Group Reference Group

Median (Range)

Total Group Reference Group

Head (%)Neck and midpiece (%)Tail (%)Cytoplasmic droplets (%)TZI†

85.8 (8.0)36.9 (10.7)6.5 (5.1)0.9 (1.0)1.50 (0.14)

85.5 (7.8)36.9 (10.4)6.3 (4.7)0.9 (1.0)1.50 (0.13)

86.0 (66.0–99.0)36.0 (12.0–66.0)5.5 (0–34.0)1.0 (0–6.0)1.49 (1.17–2.07)

86.0 (66.0–98.0)36.0 (18.0–66.0)5.0 (0–34.0)1.0 (0–6.0)1.48 (1.28–2.07)

* Numbers are given for the total group (n 5 96) and for a reference group defined by abstinence time 2–7 days and time to pregnancy # 12 cycles(n 5 80).

† TZI indicates terazoospermia index; average number of defects per abnormal spermatozoon.

Table 3. Selected percentiles for semen parameters in partners to pregnant women*

Semen Variable

Percentiles

2.5th 5th 10th WHO Value†

Volume (mL)pHSperm concentration (106/mL)Total sperm number (106/ejaculate)Progressive motility (a and b) (%)

1.27.59.1

15.429.0

1.77.7

10.622.333.2

2.17.9

16.954.343.0

2.07.2

204050

Rapid progressive motility (a) (%)Ideal spermatozoa (%)

10.22.0

15.23.0

18.04.0

25‡

Semen variable

Percentiles

97.5th 95th 90th WHO Value†

Head defects (%)Neck and midpiece defects (%)

98.062.8

97.055.0

96.052.0

‡‡

Tail defects (%)Cytoplasmic droplets (%)TZI

16.05.01.75

15.03.01.72

11.02.01.66

‡‡‡

* Abstinence time 2–7 days and time to pregnancy # 12 cycles. a indicates rapid progressive motility; b, slow or sluggish progressive motility.† Lower limit of World Health Organization (1999) reference range.‡ No value in the World Health Organization (1999) manual.

for semen variables. An arbitrary but common conventionis to define the reference interval as the central 95% in-terval bounded by 2.5th and 97.5th percentiles. However,an asymmetric location of the interval may be more ap-propriate. This would be the case with most of the semenvariables since no pathological conditions are associatedwith the upper 2.5% outside the 95% central interval.Another size interval could also be of more clinical value.Both the 5th (MacLeod and Gold, 1951; Comhaire et al,1987) and 10th percentiles have been suggested as cutoffvalues in previous studies (Ombelet et al, 1997). The re-sults from the Norwegian fertile men in this study showthat the values based on the 5th percentile are far belowthe WHO (1999) lower limit concerning sperm concen-tration and sperm motility (Table 3). Even the 10th per-centile from this study is lower than the WHO value forsperm concentration and motility. There has been an in-creasing opinion that the WHO reference ranges shouldbe reconsidered (reviewed in van der Merve et al, 2005).However, data from different studies have indicated ad-justment in both directions. In a study of first-pregnancy

planners (Bonde et al, 1998), the probability of concep-tion increased with increasing sperm concentration up to40 3 106/mL. This value was therefore considered as athreshold between subfertile and fertile men. In anotherstudy recruiting pregnant women and their partners (Sla-ma et al, 2002), an increase in sperm concentration up to55 3 106/mL was found to influence TTP. Others havecompared semen parameters in fertile and infertile menand suggested thresholds for sperm concentration from9.0 3 106/mL to 34 3 106/mL and for motility from 20%to 52%, depending on the statistical methods used andassumptions made (Ombelet et al, 1997; Gunalp et al,2001; Guzick et al, 2001; Menkveld et al, 2001). Thisindicates the difficulties involved in extrapolating cutoffvalues from one population to another if the selectioncriteria or study design are different.

The WHO (1999) manual does not give any referencerange for normal sperm morphology but states that thevalue from the WHO (1992) should be adjusted down-ward when strict criteria are applied. In the Norwegianreference group in this study the 5th percentile for ideal

70 Journal of Andrology · January/February 2006

Table 4. Comparison of median values for semen parameters among men in couples with TTP 5 1 and TTP . 1*

Semen Variable TTP 5 1 (n 5 38) TTP . 1 (n 5 59)† P

Volume (mL)Sperm concentration (106/mL)Total sperm number (106/ejaculate)Progressive motility (a and b) (%)Rapid progressive motility (a) (%)

4.0101.5410.155.538.5

3.664.0

254.252.033.0

.903

.026

.024

.022

.058Total number of sperm with progressive motility (106)Ideal spermatozoa (%)Total number of ideal sperm (106)Head defects (%)Neck and midpiece defects (%)

238.815.057.085.035.0

143.512.029.689.038.0

.013

.034

.014

.030

.488Tail defects (%)Cytoplasmic droplets (%)

5.01.0

7.01.0

.192

.611

* TTP indicates time to pregnancy; a, rapid progressive motility; b, slow or sluggish progressive motility.† n 5 56 for the morphology evaluation.

sperm morphology is 3%. Although this value is not di-rectly comparable to those from other studies, the presentresult for sperm morphology is comparable with otherstudies in which strict criteria were used. By evaluationof semen sample from fertile men, the 5th and 10th per-centiles have been reported to be 4% and 5%, respectively(Ombelet et al, 1997), and the 10th percentile, 2% (Menk-veld et al, 2001). As for the percentage of normal sperm,reference values for the various sperm defects are notgiven in the WHO (1999) manual. Calculation of terato-zoospermia index (TZI) in this manual is an optional testand is limited to head, neck and midpiece, and tail de-fects, whereas in this study TZI also includes cytoplasmicdroplets as described in the EHSRE-NAFA manual (Kvistand Bjorndahl, 2002). A multiple anomalies index (MAI)has been shown to be associated with the probability ofconception among couples with infertility problems(Jouannet et al, 1988). Furthermore, a study of TTP andsemen parameters to partners to pregnant women showedthat MAI was strongly related to the probability of con-ception (Slama et al, 2002). However, in these studiesMAI was the mean of more than 4 defects per abnormalspermatozoon and not directly comparable to TZI in thisstudy. As far as we know, only 1 study (Menkveld et al,2001) reports cutoff values for TZI based on strict criteriaand 4 defects as in the present study. The median valuein the fertile population was 1.54 (Menkveld et al, 2001)compared to 1.49 in the total group in our study and 1.48in the reference group (Table 2). The cutoff value basedon receiver operating characteristic curve analysis be-tween fertile and subfertile populations was 1.64 or 2.09if 50% prevalence of subfertility was assumed (Menkveldet al, 2001). The 95th and 90th percentiles in our studywere 1.72 and 1.66, respectively (Table 3).

The proportion of couples conceiving at the first cycle(39%) is high, which may be due to a selection of coupleswith high fertility. It was of interest to examine if thesemen parameters in men of these couples differed from

the others. Overall, the semen parameters were more fa-vorable in the TTP 5 1 group than in the TTP .1 group.There were significant differences in sperm concentration,progressive motility, and proportion of ideal spermatozoabetween groups of men categorized into TTP 5 1 andTTP . 1. It is possible to compare the groups TTP 5 1and TTP.1 with respect to semen parameters while cor-recting for potential confounders, age, and abstinence, us-ing multiple linear regression. This analysis leads to onlyminor alterations in semen parameters estimates and tothe same conclusions as far as P values are concerned.However, on the basis of multiple logistic regression anal-ysis, only the variable ‘‘total number of sperm with pro-gressive motility’’ remained in the model (P 5 .002). Al-though we compare 2 groups of fertile men, and no sub-fertile group was studied, this is in accordance with find-ings that differences between fertile and subfertilepopulations become more pronounced when semen char-acteristics are combined than when looking only at singleparameters (Bartoov et al, 1993; Ombelet et al, 1997).However, the limited sample size of the 2 fertile groupsmakes it difficult to draw any conclusion from our resultsas to which semen parameter is the best predictor of fer-tility potential.

In conclusion, estimated thresholds for the various se-men parameters to discriminate between fertile and sub-fertile men depend on the statistical methods used andpopulations studied. As recommended by WHO (1999),each laboratory should establish its own reference rangesfor the semen variables, and we estimated 5th and 10thpercentiles of semen parameters in a group of men whohad recently achieved a pregnancy. Most of these valueswere below the WHO lower limit. We suggest that the5th percentiles of the semen parameters be used for com-parison of the patients’ results, but that also the 10th per-centiles be included in the sample record form sent to thereferring physician. Table 5 shows the reference ranges

71Haugen et al · Semen Parameters in Fertile Men

Table 5. Reference ranges for semen variables established for theAndrology Laboratory in Oslo*

Volume (mL)pHSperm concentration (106/mL)Total sperm number (106/ejaculate)Progressive motility (a and b) (%)

$1.7$7.7$11$22$33

Rapid progressive motility (a) (%)Ideal spermatozoa (%)

$15$3

* a indicates rapid growth progressive motility; b, slow or sluggish pro-gressive motility.

for semen variables established for the Andrology Labo-ratory in Oslo on the basis of the 5th percentiles.

AcknowledgmentsWe thank Aase Strutz and Trine Henrichsen for performing the semenanalyses.

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